This invention is concerned with a reagent composition and method for the detection of peroxidase activity. The invention is particularly concerned with the detection of pseudo-peroxidase activity of hemoglobin in occult blood present in fecal matter and other biological specimens.
Colon and rectal cancers affect over 100,000 persons in the United States each year. Early diagnosis and treatment of these cancers as well as cancers of other digestive organs can result in a cure rate of 80% to 90% of those affected. However, late diagnosis and treatment reduces the cure rate to 25% or less. Thus early detection of this disease in the digestive tract is critical to its successful treatment.
Most cancers of the digestive tract bleed to a certain extent even in their early stages giving rise to occult or hidden blood in fecal matter. It has been the goal of many researchers to develop methods and tests for determining the presence of this occult blood by detecting the peroxidase-like activity of hemoglobin in the blood. One of the most widely used indicator reagents for diagnosing occult blood is derived from an extract from the wood of certain species of trees of the Guaiacum genus native to the American tropics. The extract, termed guaiac, turns from essentially colorless to blue in the presence of hemoglobin and an oxidizing agent such as hydrogen peroxide.
The tests most widely used today can be divided into two basic categories: the "direct contact" type and the "in the bowl" type. In the "direct contact" type a small sample of the feces is contacted with a chromophoric reagent deposited on some form of carrier and it is observed if the reagent undergoes a color change. In the "in the bowl" type, the reagent(s) on a support or carrier is placed in the toilet water containing fecal matter to be tested and any color change is observed. An example of the first "direct contact" type is the "Hemoccult" test disclosed in U.S. Pat. No. 3,966,006 and marketed by Smith Kline Diagnostics of Sunnyvale, Calif. Briefly, the test employs an adsorbent white paper impregnated with a guaiac reagent encased in a special test slide having openable flaps on both sides of the test slide. To use the test slide, one must obtain a sample of fecal matter, smear it onto the guaiac-impregnated paper by opening the panel on one side of the test slide, and thereafter closing the panel. A panel on the opposite side of the test slide is then opened and a developing agent, which is a stabilized solution of hydrogen peroxide and denatured alcohol, is applied to the guaiac-impregnated paper. If occult blood is present in the fecal matter smeared on the opposite side of the paper, the product of the guaiac reaction will appear as a blue substance against the white paper background, providing a positive indication of the presence of blood in the fecal matter.
Although the test is suitable for use by physicians in their offices and by diagnostic laboratories, it is not the type of test that is readily adaptable for use by the ordinary person because of their adverse reaction to handling fecal matter and because of lack of skill in interpreting the results. As stated above, the test requires that a specimen of fecal matter be obtained. Normally, a specimen is obtained by procuring a sample on the end of a spatula or a wooden tongue depressor, which is then used to smear the specimen on the paper in the test slide. Once the sample is obtained and the test procedure completed, both the test slide and the spatula or depressor must be disposed of. Disposal of the used materials can and does present a physical problem to, if not an adverse psychological reaction for, the ordinary person. Thus, the ordinary person is not likely to use the test because of the uncleanly nature and because of the disposal problems associated with the used test slide and spatula or depressor. Additionally, the ordinary person does not necessarily have the skill required to analyze, and thus form accurate conclusions from the test results.
As an alternative, the ordinary person can initiate the test in his home and then forward the test slide to his physician or a laboratory for addition of the developing agent and analysis of the test. This procedure, however, requires cold storage of the test slide and specimen if there is a significant time lapse before the test can be completed. Certainly, the ordinary person does not wish to store a fecal specimen in his household refrigerator, normally the only cold storage available to him, until he can present the specimen to his physician or an appropriate laboratory. Thus, the general public is not likely to follow or comply with this alternative.
U.S. Pat. No. 4,473,079 provides a direct contact test for occult blood in feces including an oxidation reduction indicator such as gum guaiac or benzidine carried by a support, a solid oxygen releasing compound such as sodium perborate on a spreadable medium and an applicator for sampling the biological substance to be tested.
An "in-the-bowl" test for occult blood is disclosed in U.S. Pat. No. 2,838,377 to D. E. Fonner. The basic test reagents employed by Fonner are o-toluidine and benzidine. These reagents, in the presence of blood and other reactants, produce a dye visible to the naked eye. Although the Fonner test appears to be a solution to the problem of finding a viable home test for occult blood, it has not met with success for two reasons. First, the above-listed reagents are in themselves known to cause cancer and thus are not suitable for general public distribution. More importantly, the Fonner reagents have a relatively high rate of providing false indications of the presence of occult blood as a result of tap water impurities such as metals.
In U.S. Pat. No. 4,385,114 to Werner Guthlein et al there is provided a process for the detection and determination of peroxidatively active substances by contacting a sample with a 3,3',5,5'-tetraalkylbenzidine and hydrogen peroxide or a substance which reacts with hydrogen peroxide and evaluating the coloration production by the reaction. The method is applied particularly for the detection of blood and glucose in urine and for cholesterol and cholesterol esters in serum. The method is specifically applied by successively impregnating filter paper with a solution containing the peroxide component and a solution containing the benzidine compound. After drying, the test paper is dipped into a urine sample and any color change, e.g. blue-green is noted.
In U.S. Pat. No. 4,175,923 to W. G. Friend an in-the-bowl occult blood test is described in which the user must apply a hydrogen peroxide solution to a sheet of paper impregnated with gum guaiac. The sheet is then placed into the toilet bowl and observed for a color change. In U.S. Pat. No. 4,541,987 to Guadagno a granular chemical formulation of a guaiacol sulfonic acid and a monopersulfate is sandwiched in pockets between an adsorbent paper and a non-adsorbent paper. The product is used by laying the assembly on the surface of the toilet bowl water, adsorbent paper facing down, and observing any color changes that take place.
The present invention, on the other hand, provides a dry, granular and stable reagent for the detection of occult blood in fecal matter in aqueous media, i.e. "in the bowl" in which all reactive components are mixed together and no reaction takes place until the reagent is wetted by the water. A sequesterant for the heavy metals found in household water supplies may also be included.